1. Field of the Invention
The present invention generally relates to a novel antibody against polyethylene glycol (PEG) and a process of using this novel antibody to accelerate the clearance of polyethylene glycol conjugates in the human body in cancer therapy as well as a method to quantify the concentration of PEG modified compounds.
2. Description of the Related Art
Recombinant proteins are increasingly being employed for the therapy of a wide variety of diseases (Nemunaitis, 1997; Fareed et al., 1998; Hudson, 1998; Lin, 1998; Harker, 1999; Sandborn and Hanauer, 1999). Many of the proteins in clinical development are modified by the covalent attachment of methoxypoly(ethylene glycol) (PEG) (Menzel et al., 1993; Basser et al., 1996; Fareed et al., 1998; Goffin et al., 1999; Harker, 1999), a flexible, linear polymer containing repeating —[OCH2CH2]— subunits (Delgado et al., 1992). PEG-modified proteins often exhibit prolonged circulation half-lives (Beckman et al., 1988; Cheng et al., 1997; Chapman et al., 1999) and reduced proteolytic cleavage (Roseng et al., 1992; Kaneda et al., 1995; Brinckerhoff et al., 1999). Immune responses against proteins can also be decreased by covalent attachment of PEG (Abuchowski et al., 1977a). Reduction of immunogenicity can be an important consideration because even recombinant human proteins can induce a humoral immune response (Atkins et al., 1986; Gribben et al., 1990). Conjugates formed between drugs and PEG have also recently been developed (Caliceti et al., 1993; Conover et al., 1997; Greenwald et al., 1998; Pendri et al., 1998). In addition, covalent attachment of PEG to liposomes has been found to reduce non-specific uptake as well as increase liposome stability and half-life (Kirpotin et al., 1997; Cabanes et al., 1998; Meyer et al., 1998).
Clinical development of PEG-modified proteins requires measurement of the pharmacokinetics in animals and patients. Ideally, the concentration of intact PEG-modified protein should be measured. Simple methods to measure intact PEG-protein conjugates, however, are not available. Analysis of PEG-modified proteins has been problematic (Delgado et al., 1992). Sodium dodecylsulfate polyacrylamide gel electrophoresis can be used to measure the relative size of PEG-modified proteins, but the mobility of PEG-modified proteins is slower than the expected molecular weight (Suzuki et al., 1984; Katre et al., 1987). Conjugates can be indirectly measured by first radiolabeling the protein (Kaneda et al., 1995; Cheng et al., 1997; Yabe et al., 1999) or PEG (Mullin et al., 1997), but radioisotopes pose safety concerns and require special handling. Functional assays can be employed to measure the concentration of the protein component of conjugates (Cheng et al., 1997; Esslinger et al., 1997), but no information is provided about the stability of covalently attached PEG chains. Methods that measure the number of PEG molecules attached to a protein (Habeeb, 1966; Stocks et al., 1986) require that purified conjugate be employed which is difficult to achieve in pharmacokinetic studies. Methods that directly measure the concentration of PEG are relatively insensitive. Colorimetric methods based on complex formation between barium-iodide and PEG require that proteins are first removed and have detection limits of around 1-5 μg PEG (Childs, 1975). A colorimetric method based on partitioning of a chromophore present in aqueous ammonium ferrothiocyanate reagent can be employed for complex protein mixtures but has a detection limit of 1-5 μg PEG (Nag et al., 1996; Nag et al., 1997). High performance liquid chromatography can detect PEG with a detection limit around 1-5 μg/ml (Kinahan and Smyth, 1991; Ryan et al., 1992; Ruddy and Hadzija, 1994; Miles et al., 1997). Phase-partitioning can be employed to measure PEG but the assay sensitivity is about 1 μg PEG (Guermant et al., 1995). Finally, polyclonal antibodies against PEG can detect the presence of 1 μg/ml PEG in PEG-modified proteins (Richter and Akerblom, 1983).
The development of monoclonal antibodies against PEG could allow quantitation of PEG and PEG-modified proteins by standard immunoligical assays including ELISA, immunoblotting, dot blotting and radioimmunoassay. It is difficult, however, to produce antibodies against PEG due to its ability to modulate immune responses. For example, immune responses against proteins are often decreased by covalent attachment of PEG (Abuchowski et al., 1977b). PEG modification has been shown to reduce the immunogenicity of enzymes (Abuchowski et al., 1977a; Chaffee et al., 1992), antibodies (Kitamura et al., 1991), toxins (Wang et al., 1993; He et al., 1999), recombinant human proteins (Katre, 1990) and other proteins (Chinol et al., 1998). PEG-modified proteins often induce tolerance to the unmodified protein in many (Lee et al., 1981; Savoca et al., 1984; Maiti et al., 1988; Saito et al., 1996; Ito et al., 1997; Ito et al., 1998), but not all cases (Savoca et al. 1979; Chen et al., 1981). Rabbit polyclonal antibodies against PEG have been generated by immunizing rabbits with PEG linked to different proteins (Richter and Akerblom, 1983). PEG alone did not generate an immune response. PEG linked to bovine superoxide dismutase or ragweed pollen extract did not reproducibly generate an anti-PEG response even when the imunogens were given in Freund's complete adjuvant. PEG-modified ovalbumin in Freund's complete adjuvant produced an antibody response in most but not all animals. Antisera raised against PEG-modified ovalbumin could detect PEG-modified proteins with a limit of detection of approximately 1 μg/ml (Richter and Akerblom, 1983). However, no monoclonal antibodies have been successfully produced against PEG to date. Monoclonal antibodies possess advantages compared to polyclonal anti-serum for standardized assays including the ability to produce unlimited quantities of homogeneous antibody with consistent binding affinity.
A major goal of anti-tumor drug development is to increase the therapeutic index of chemotherapy, thereby improving treatment efficacy. One approach to increase the therapeutic index of chemotherapy is to preferentially activate antineoplastic prodrugs at cancer cells but not normal tissues. Tumor selectivity may be achieved by enzymatically converting prodrugs possessing low toxicity to highly toxic anti-neoplastic agents by previously administered antibody-enzyme conjugates (immunoenzymes) that have been allowed to accumulate at tumor cells (Bagshawe et al., 1988; Senter et al., 1988). Even though maximum accumulation of immunoenzymes in tumors occurs around 24 h after administration (Bosslet et al., 1994; Wallace et al., 1994), prodrugs are generally administered from 3-7 days (Bosslet et al., 1994; Svensson et al., 1998) to up to 2 weeks (Eccles et al., 1994) later to allow adequate time for conjugate to clear from the blood, thereby minimizing systemic prodrug activation and associated toxicity to normal tissues. The requisite of low circulating levels of immunoenzyme therefore often precludes prodrug administration when maximum localization has been achieved.
Prodrugs can be administered during the period of maximum tumor accumulation of immunoenzymes if circulating conjugates are removed or deactivated. Several methods have been devised to accelerate the clearance of radioimmunoconjugates and immunoenzymes from the circulation including the administration of polyclonal (Stewart et al., 1990) and anti-idiotypic antibodies (Ullen et al., 1995a; Ullen et al., 1995b) against the antibody portion of the immunoconjugate, injection of avidin to clear biotinylated antibodies (Klibanov et al., 1988; Paganelli et al., 1991; Stella et al., 1994; Kobayashi et al. 1995), use of monoclonal antibodies against enzymes to clear (Kerr et al., 1993; Haisma et al., 1995) or deactivate (Sharma et al., 1990) immunoenzymes, and extracorporeal immunoadsorption (Tennvall et al., 1997) to remove immunoconjugates from plasma.
β-Glucuronidase targeted to tumor cells can activate the glucuronide prodrug (BHAMG) of p-hydroxy aniline mustard (pHAM) in vitro (Wang et al., 1992) and cure advanced hepatoma ascites in a rat model (Chen et al., 1997). Antibody-βG conjugates injected intravenously, however, are rapidly cleared from the circulation before significant tumor accumulation occurs (Cheng et al., 1997). Modification of βG with PEG can extend the half-life of antibody-βG conjugates, decrease the normal tissue uptake, and increase the localization of the conjugate at the solid tumors in nude mice (Cheng et al., 1997). Although the extended half-life of PEG-modified βG conjugate is desirable for improved tumor uptake, at least five days were required for the serum concentration of the conjugates to reach a safe level before a prodrug could be administered (Cheng et al., 1997). The accelerated clearance of conjugates from the circulation may allow earlier prodrug administration when the maximum amount of the conjugate is present at tumor cells. We generated mAbs against βG and PEG and examined their effects on the clearance of the βG-sPEG conjugates. The effect of incorporating galactose residues in the clearing antibodies was also examined because galactose can accelerate the removal of proteins from the circulation (Thornburg et al., 1980) by the hepatic asialoglycoprotein receptor (Ong et al., 1991). Rapid clearance of an antibody-carboxypeptidase G2 conjugate by a galactose-modified antibody (Sharma et al., 1990; Sharma et al., 1994) allowed earlier administration of a prodrug with reduced toxicity (Rogers et al., 1995). In addition, galactose-modified mAb has also been employed in a clinical trial to remove residual antibody-carboxypeptidase G2 conjugate from the circulation before prodrug administration (Martin et al., 1997).